Novel compound heterozygous mutations in MYO7A in a Chinese family with Usher syndrome type 1

Purpose To identify the disease-causing mutation(s) in a Chinese family with autosomal recessive Usher syndrome type 1 (USH1). Methods An ophthalmic examination and an audiometric test were conducted to ascertain the phenotype of two affected siblings. The microsatellite marker D11S937, which is close to the candidate gene MYO7A (USH1B locus), was selected for genotyping. From the DNA of the proband, all coding exons and exon-intron boundaries of MYO7A were sequenced to identify the disease-causing mutation(s). Restriction fragment length polymorphism (RFLP) analysis was performed to exclude the alternative conclusion that the mutations are non-pathogenic rare polymorphisms. Results Based on severe hearing impairment, unintelligible speech, and retinitis pigmentosa, a clinical diagnosis of Usher syndrome type 1 was made. The genotyping results did not exclude the USH1B locus, which suggested that the MYO7A gene was likely the gene associated with the disease-causing mutation(s) in the family. With direct DNA sequencing of MYO7A, two novel compound heterozygous mutations (c.3742G>A and c.6051+1G>A) of MYO7A were identified in the proband. DNA sequence analysis and RFLP analysis of other family members showed that the mutations cosegregated with the disease. Unaffected members, including the parents, uncle, and sister of the proband, carry only one of the two mutations. The mutations were not present in the controls (100 normal Chinese subjects=200 chromosomes) according to the RFLP analysis. Conclusions In this study, we identified two novel mutations, c.3742G>A (p.E1248K) and c.6051+1G>A (donor splice site mutation in intron 44), of MYO7A in a Chinese non-consanguineous family with USH1. The mutations cosegregated with the disease and most likely cause the phenotype in the two affected siblings who carry these mutations compound heterozygously. Our finding expands the mutational spectrum of MYO7A.

vestibular function may also be impaired, which causes a delay in motor development and walking in those affected compared to normal children. Onset of RP often occurs in the first decade of life, with a progressively constricted visual field and impaired visual acuity. In USH1 families, MYO7A is the most commonly mutated gene, accounting for approximately 50% [14,15], followed by CDH23, PCDH15, USH1C, and USH1G.
The MYO7A gene is located on the long arm of chromosome 11 at position 13.5 (11q13.5). The biggest transcript of MYO7A consists of 49 exons and encodes a 2,215 amino acid unconventional myosin named myosin VIIA. Thus far, more than 250 MYO7A mutations have been reported. Most of these mutations (more than 95%) cause Usher syndrome type 1, while the rest of the mutations are responsible for autosomal recessive or dominant non-syndromic deafness, according to the Human Gene Mutation Database Professional 2012.2.
Here we report on the investigation of a Chinese family with early onset of combined blindness and deafness, key features of USH1. Genotyping results suggested that MYO7A was likely the gene associated with the disease in this family. Through direct DNA sequence analysis of the 48 coding exons of MYO7A, we identified two novel compound heterozygous mutations that likely cause the observed phenotype.

Study subjects:
A Chinese family consisting of 11 individuals (seven participated in this study) from Hubei province was investigated in this study. Written informed consent was obtained from the participants, and the research was approved by the Ethics Committee of Huazhong University of Science and Technology. Detailed ophthalmologic examinations of the subjects, including visual acuity, slit-lamp biomicroscopy, applanation intraocular pressure, visual field, dilated funduscopic examination, and fundus photography, as well as audiometric tests, were performed at the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Whole peripheral blood samples were collected by clinicians in Venous Blood Vacuum Collection Tubes containing tripotassium EDTA as anticoagulant, from seven family members in the Union Hospital, Tongji Medical College, Wuhan, PRC. The blood samples were stored at -4 °C for up to 7 days before DNA was extracted using the DNA isolation kit for mammalian blood (Tiangen Biotech Co., Ltd., Beijing, China).
Genotyping: To date, six genes associated with USH1 have been cloned. Haplotype analysis was done in the order of their causal frequency: MYO7A, CDH23, PCDH15, USH1C, USH1G and CIB2 [8,16]. For MYO7A, the microsatellite marker D11S937 (77.8 Mb on chromosome 11, Applied Biosystems, Inc., Foster City, CA), which is tightly linked to the candidate gene MYO7A (76.8 Mb on chromosome 11), was selected for genotyping. The PCR products were separated by electrophoresis on an ABI 3100 Genetic Analyzer (Applied Biosystems), and genotypes were determined with Peak Scanner Software v1.0 (Applied Biosystems). Digested PCR products were run on 1.5% agarose gels. Samples from all available family members and 100 normal controls were used in the RFLP analysis.

RESULTS
Clinical examinations and pedigree analysis: There are 11 individuals in the non-consanguineous family ( Figure 1). The proband's paternal grandparents and maternal grandparents were not enrolled in the genetic study. The 20-year-old proband had gradually declining night vision and hearing loss since childhood and so did his younger brother. The proband was emmetropic with Snellen visual acuity recorded as 0.6 in both eyes. Examination of the anterior segment showed normal cornea, clear anterior chamber, and transparent lens. Fundus photography revealed vessel attenuation and characteristic bone spicule-like pigment with macular involvement (Figure 2). Pure tone audiometry showed severe to profound bilateral sensorineural hearing impairment ( Figure 3). The two affected individuals have completely lost their hearing and are practically unable to speak. Other members of the family are normal. According to the family history and results from the audiometric test and ophthalmic examinations, we concluded that the two patients display the typical presentation of autosomal recessive Usher syndrome type 1 (Figure 1).
Genotyping and mutational analysis: The genotyping results did not exclude MYO7A, suggesting that one or more mutation(s) in MYO7A likely caused the phenotype observed in the two patients. Sequence analysis of all 48 coding exons and exon-intron boundaries of MYO7A revealed in the proband two novel compound heterozygous mutations. One was a c.3742G>A mutation in exon 29, which resulted in a substitution of lysine for glutamic acid at codon 1248 (p.E1248K; Figure 4A). The other was a G to A change at the conserved donor splice site in intron 44 (c.6051+1G>A; Figure 4B). We performed DNA sequencing for other available family members. The results indicated that the two mutations completely cosegregated with the disease in this family.
We further performed RFLP analysis for the family members as well as other 100 normal individuals. To identify the mutation c.3742G>A, a 454-bp fragment was amplified and digested by BstXI. As shown in Figure 4C, for homozygous normal individuals II1, II3, and II4, the restrictive digestion by BstXI resulted in two bands of 282 bp and 172 bp. In contrast, for the heterozygous individuals (II2, III1, III2, and III3) who carry the mutation c.3742G>A, the restrictive digestion by BstXI resulted in three bands of 454 bp, 282 bp, and 172 bp.
To identify the mutation c.6051+1G>A, a 99-bp fragment was amplified and digested by ScrFI. As shown in Figure  4D, for homozygous normal individuals II1, II2, and III3, the restrictive digestion resulted in a band of about 50 bp. In contrast, for the heterozygous individuals (II3, II4, III1, and III2) who carry the mutation c.6051+1G>A, the restrictive digestion resulted in two bands of 50 bp and 99 bp. These results confirmed that our patients III1 and III2 with USH carry both mutations of c.3742G>A and c.6501+1G>A. RFLP analysis also indicated that these two mutations were not present in the 100 normal control individuals. The RFLP results for some of the normal controls are shown in Figure 4E.    (1747-2205). The motor domain is also known as the myosin head-like domain, and could bind F-actin and ATP [17].
Myosin VIIA is expressed in the hair cells of the inner ear, the retinal pigment epithelium (RPE), and the photoreceptor cells of the retina. In the photoreceptor cells, myosin VIIA is present at the connecting cilium and regulates opsin transport [18]. In the RPE cells, myosin VIIA participates in the light cycle-dependent movement of melanosome transportation [19] and the normal functioning of the visual retinoid cycle [20] and is associated with lysosomes [21]. In the hair cells of the inner ear, myosin VIIA and the other four USH1-related proteins (harmonin encoded by USH1C, Sans encoded by USH1G, CDH23 encoded by USH1D, PCDH15 encoded by USH1F) participate in the formation of the mechanotransduction complex [22], which is critical for detecting sound.
Several proteins have been identified to interact with myosin VIIA. In addition to interacting with actin, harmonin, Sans, CDH23, and PCDH15, myosin VIIA also interacts with MYRIP [19], RPE65 [20], and WHRN (USH2D) [23]. Myosin VIIA is predominantly monomeric in cells, but can be induced to dimerize via the predicted coiled-coil-domain after cargo binding and functions as a cargo transporter [24].
The c.3742G>A mutation was predicted to cause a glutamic acid change to lysine at the highly conserved codon 1248 in the first MyTH4-FERM tandem domain, which mediates the interaction between myosin VIIA and the scaffold protein Sans (USH1G) [25]. The E1248K mutation may prevent this interaction and thus could disrupt the normal function of myosin VIIA.
The c.6051+1G>A mutation is a donor splice site mutation in intron 44 of the MYO7A gene. Using the online software NetGene2 v2.4, we predicted an impact of the mutation on pre-mRNA splicing of MYO7A. The mutation would destroy that splice site, leading to a truncated protein lacking the last approximate 200 amino acid residues. The C-terminal tail of myosin VIIA, containing the SH3, MyTH4, and FERM domains (1605-2215), interacts with harmonin (USH1C) [26]. Therefore, the mutant myosin VIIA protein may lose the ability to bind harmonin, which acts as a scaffold protein linking the proteins in the cell membrane to the proteins in the cytoskeleton. The additive effects of the two identified compound heterozygous mutations could cause a complete dysfunction of myosin VIIA in the two patients, leading to the observed phenotype.
Applying genotyping microarray and next-generation sequencing will allow for large-scale screening in patients with USH. These methods will become the standard approaches in clinical diagnosis and scientific research in the future. Although numerous mutations in USH genes  have been found, much remains to be done to understand the pathogenesis of Usher syndrome and facilitate development of potential treatments for the disease.